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COMPARATIVE STUDY OF THE ANTIBACTERIAL PROPERTIES OFASCORBIC ACID AND REDUCTOGENIC COMPOUNDS'QUENTIN N. MYRVIK AND WESLEY A. VOLKDepartment of Microbiology, School of Medicine, University of Virginia, Charlottesville, VirginiaReceived for publication May 24, 1954Ascorbic acid has an antibacterial effectagainst tubercle bacilli (Boievain and Spillane,1937; Leitner, 1937; Sirsi, 1952) and many othermicroorganisms(von Gagyi, 1936; Grootten andBezssonoff, 1935; Lwoff and Morel, 1942a;Ehrismann, 1942). The mechanism of action andthe chemical group responsible for the bacterialinhibition are not known.Bactericidal effect due to a lowering of the pHhas been suggested by von Gagyi (1936) andEhrismann (1942); however, Slade and Knox(1950) have found ascorbic acid to be bac-teriostatic for a group A hemolytic streptococcuseven though the pH was near neutrality. Ehris-mann (1942) found that anaerobes were generallystimulated by ascorbic acid, whereas strict aer-obes were generally inhibited, suggesting that inthe latter inhibition was due to a reduction inthe O/R potential of the medium. Lwoff andMorel (1942b) found that inhibition of Proteusvulgaris by ascorbic acid was counteracted by thepresence of reducing agents and by substanceswhich catalyzed the breakdown of hydrogen per-oxide. Simiar results were obtained with Escher-ichia coli and Bacilus subtils. They concludedthat the inhibition was due to hydrogen peroxideformed during the auto-oxidation of ascorbic acid.Myrvik et al. (1954) reported that some auto-oxidized derivative of ascorbic acid was mostlikely the active principle against mycobacteria.The addition of catalase in the form of rabbit redblood cell lysates failed to influence the inhibition.Furthermore, they were unable to asociate anyappreciable tuberculostatic activity with any ofthe classical breakdown products of ascorbic acidsuch as 2,3-diketogulonic acid, oxalic acid,furfural, and threonic acid. The active principlewas heat-stable, water soluble, and sparingly1 Aided by a medical research grant from theNational Tuberculosis Association, through itsMedical Section, the American Trudeau Society,and by a research grant, G-3591, from the NationalInstitutes of Health, Public Health Service.soluble in 95 per cent alcohol but was insolublein acetone, ether, petroleum ether, benzene,carbon tetrachloride, and chloroform.The present investigation was an attempt todetermine the chemical group responsible for theantibacterial properties of ascorbic acid solutions.Evidence is presented which indicates that theseinhibitory properties reside in the oxidizedenediol (diketone) group.TERI8 AND METHODSMicroorganisms used and source. BCG strainof Mycobacterium tuberculosis var. bovis and M.phlei: Dr. R. S. Weiser, Department of Micro-biology, University of Washington, School ofMedicine, Seattle, Washington. Ravenel strainof M. tuberculis var. bovis and H37Rv strain ofM. tuberculosis var. hominis8: Mr. W. Steenken,Jr., Trudeau Laboratory, Trudeau, New York.Escherichia coli and Psudomonas sp.: Culturecollection, Department of Microbiology, Uni-versity of Virginia, School of Medicine, Char-lottesville, Virginia.The mycobacteria were maintained at 37 Con Youmans' modification (1946) of Proskauerand Beck's broth. Inocula were prepared bygrinding a portion of the surface growth in asterile mortar and diluting with sterile saline.The other microorganisms were maintained at37 C in nutrient broth (Difco).Antibaderial tests. The antibacterial tests wereconducted using Youmans' (1946) modificationof Proskauer and Beck's medium containing 5per cent Seitz filtered bovine serum. The com-pounds to be tested were disolved in distilledwater, adjusted to pH 7.0, sterilized by filtrationthrough a Swinny filter, and serially doublediluted in one ml amounts of the above medium.Each tube was inoculated with one drop of abacterial suspension containing approximately10 3mg of wet weight standardized in a Colemannephelometer model 9. Cultures of the slowgrowing mycobacteria were incubated at 37 C622